Protease variants and compositions

ABSTRACT

The present invention relates to enzymes produced by mutating the genes for a number of subtilases and expressing the mutated genes in suitable hosts are presented. The enzymes exhibit improved wash performance in any detergent in comparison to their wild type parent enzymes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/336,324 filed Jan. 3, 2003, now U.S. Pat No. 6,921,657, which is acontinuation of U.S. application Ser. No. 09/512,251 filed Feb. 24,2000, now U.S. Pat. No. 6,555,355, which is a continuation ofinternational application no. PCT/DK98/00359 filed Aug. 19, 1998, whichclaims priority under 35 U.S.C. 119 of Danish application no. 0986/97filed Aug. 29, 1997, the contents of which are fully incorporated hereinby reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to novel mutant protease enzymes or enzymevariants useful in formulating detergent compositions and exhibitingimproved wash performance in detergents; cleaning and detergentcompositions containing said enzymes; mutated genes coding for theexpression of said enzymes when inserted into a suitable host cell ororganism; and such host cells transformed therewith and capable ofexpressing said enzyme variants.

2. Description of Related Art

In the detergent industry enzymes have been implemented in washingformulations for more than 30 years. Enzymes used in such formulationscomprise proteases, lipases, amylases, cellulases, as well as otherenzymes, or mixtures thereof. Commercially most important enzymes areproteases.

An increasing number of commercially used proteases are proteinengineered variants of naturally occurring wild type proteases, e.g.,DURAZYM® (Novo Nordisk A/S), RELASE® (Novo Nordisk A/S), MAXAPEM®(Gist-Brocades N.V.), PURAFECT® (Genencor International, Inc.).

Further a number of protease variants are describe in the art, such asin EP 130756 (Genentech) (corresponding to U.S. Reissue Pat. No. 34,606(Genencor)); EP 214435 (Henkel); WO 87/04461 (Amgen); WO 87/05050(Genex); EP 260105 (Genencor); Thomas, Russell, and Fersht, Nature 318:375-376 (1985); Thomas, Russell, and Fersht, J. Mol. Biol. 193: 803-813(1987); Russel and Fersht, Nature 328: 496-500 (1987); WO 88/08028(Genex); WO 88/08033 (Amgen); WO 95/27049 (Solvay S.A.); WO 95/30011(Procter & Gamble Company); WO 95/30010 (Procter & Gamble Company); WO95/29979 (Procter & Gamble Company); U.S. Pat. No. 5,543,302 (SolvayS.A.); EP 251 446 (Genencor); WO 89/06279 (Novo Nordisk A/S); WO91/00345 (Novo Nordisk A/S); EP 525 610 A1 (Solvay); WO 94/02618(Gist-Brocades N.V.); and WO 96/34946 (Novo Nordisk A/S).

However, even though a number of useful protease variants have beendescribed, there is still a need for new improved protease variants fora number of industrial uses.

Therefore, an object of the present invention is to provide improvedprotein engineered protease variants, especially for use in thedetergent industry.

SUMMARY OF THE INVENTION

The present inventors have intensively studied numerous of the possiblecombinations of the N252, T255 and S259 residues of SAVINASE®, andidentified a number of variants with increased improved washperformance.

Accordingly, the present invention relates in its first aspect to asubtilase protease variant having improved wash performance indetergents, comprising modification(s) at position(s) 252, 255 and/or259.

Preferably a subtilase variant according to the invention comprisesmodifications in both positions 252 and 255, and more preferredcomprises modifications in all three positions 252, 255, and 259.

In a second aspect the invention relates to a subtilase enzyme varianthaving improved wash performance in detergents, comprising at least onemodification chosen from the group comprising:

252L+255I

252V+255A

252M+255C+259H

252S+255E+259C

252K+255S+259C; or

a variant comprising one or more conservative modification(s) in any ofthe above mentioned variants (e.g., a conservative modification of a252L (a hydrophobic amino acid)+255I includes variants such as 252I (ahydrophobic amino acid)+255I, and 252V (a hydrophobic amino acid)+255I.

In a third aspect the invention relates to an isolated DNA sequenceencoding a subtilase variant of the invention.

In a fourth aspect the invention relates to an expression vectorcomprising an isolated DNA sequence encoding a subtilase variant of theinvention.

In a fifth aspect the invention relates to a microbial host celltransformed with an expression vector according to the fourth aspect.

In a further aspect the invention relates to the production of thesubtilisin enzymes of the invention by inserting an expression vectoraccording to the fourth aspect into a suitable microbial host,cultivating the host to express the desired subtilase enzyme, andrecovering the enzyme product.

Even further the invention relates to a composition comprising asubtilase variant of the invention.

Finally the invention relates to the use of the mutant enzymes for anumber of industrial relevant uses, in particular for use in cleaningcompositions and cleaning compositions comprising the mutant enzymes,especially detergent compositions comprising the mutant subtilisinenzymes.

Definitions

Prior to discussing this invention in further detail, the followingterms will be defined.

Nomenclature of Amino Acids A = Ala = Alanine V = Val = Valine L = Leu =Leucine I = Ile = Isoleucine P = Pro = Proline F = Phe = Phenylalanine W= Trp = Tryptophan M = Met = Methionine G = Gly = Glycine S = Ser =Serine T = Thr = Threonine C = Cys = Cysteine Y = Tyr = Tyrosine N = Asn= Asparagine Q = Gln = Glutamine D = Asp = Aspartic Acid E = Glu =Glutamic Acid K = Lys = Lysine R = Arg = Arginine H = His = Histidine X= Xaa = Any amino acid

Nomenclature of Nucleic Acids A = Adenine G = Guanine C = Cytosine T =Thymine (only in DNA) U = Uracil (only in RNA)Nomenclature of Variants

In describing the various enzyme variants produced or contemplatedaccording to the invention, the following nomenclatures have beenadapted for ease of reference:

Original Amino Acid Position Substituted Amino Acid

Thus, the substitution of glutamic acid for glycine at position 195 isdesignated as:Gly 195 Glu or G195E,a deletion of glycine at the same position is designated:Gly 195* or G195*and an insertion of an additional amino acid residue such as lysine isdesignated:Gly 195 GlyLys or G195GK

Where a deletion in comparison with the sequence used for the numberingis indicated, an insertion at such a position is indicated as:*36 Asp or *36Dfor an insertion of aspartic acid at position 36.

Multiple mutations are separated by pluses, i.e.,:Arg 170 Tyr+Gly 195 Glu or R170Y+G195Erepresenting mutations at positions 170 and 195 substituting tyrosineand glutamic acid for arginine and glycine, respectively.Proteases

Enzymes cleaving the amide linkages in protein substrates are classifiedas proteases, or (interchangeably) peptidases (see Walsh, 1979,Enzymatic Reaction Mechanisms. W. H. Freeman and Company, San Francisco,Chapter 3).

Numbering of Amino Acid Positions/Residues

For purposes of the invention, the numbering of amino acids correspondsto that of the amino acid sequence of subtilase BPN′ (BASBPN), unlessotherwise indicated. For further description of the subtilisin BPN′sequence, see Siezen et al., Protein Engng. 4 (1991) 719-737 and FIGS.1A and 1B.

Serine Proteases

A serine protease is an enzyme which catalyzes the hydrolysis of peptidebonds, in which there is an essential serine residue at the active site(White, Handler and Smith, 1973 “Principles of Biochemistry,” FifthEdition, McGraw-Hill Book Company, NY, pp. 271-272).

The bacterial serine proteases have molecular weights in the range of20,000 to 45,000 daltons. They are inhibited bydiisopropylfluorophosphate. They hydrolyze simple terminal esters andare similar in activity to eukaryotic chymotrypsin, also a serineprotease. A more narrow term, alkaline protease, covering a subgroup,reflects the high pH optimum of some of the serine proteases, from pH9.0 to 11.0 (for review, see Priest (1977) Bacteriological Rev. 41711-753).

Subtilases

A sub-group of the serine proteases tentatively designated subtilaseshas been proposed by Siezen et al., Protein Engng. 4: 719-737 (1991).They are defined by homology analysis of more than 40 amino acidsequences of serine proteases previously referred to as subtilisin-likeproteases. A subtilisin was previously defined as a serine proteaseproduced by Gram-positive bacteria or fungi, and according to Siezen etal. now is a subgroup of the subtilases. A wide variety of subtilaseshas been identified, and the amino acid sequence of a number ofsubtilases has been determined. For a more detailed description of suchsubtilases and their amino acid sequences reference is made to Siezen etal. and FIGS. 1A and 1B.

One subgroup of the subtilases, I-S1, comprises the “classical”subtilisins, such as subtilisin 168, subtilisin BPN′, subtilisinCarlsberg (ALCALASE®, NOVO NORDISK A/S), and subtilisin DY.

A further subgroup of the subtilases I-S2, is recognized by Siezen etal. (supra). Sub-group I-S2 proteases are described as highly alkalinesubtilisins and comprise enzymes such as subtilisin PB92 (MAXACAL®,Gist-Brocades NV), subtilisin 309 (SAVINASE®, NOVO NORDISK A/S),subtilisin 147 (ESPERASE®, NOVO NORDISK A/S), and alkaline elastase YaB.

“SAVINASE®”

SAVINASE® is subtilisin 309 from B. lentus and differs from BABP92 onlyin having N87S (see FIGS. 1A and 1B), and is marketed by NOVO NORDISKA/S.

Parent Subtilases

The term “parent subtilase” is a subtilase defined according to Siezenet al. (Protein Engineering 4:719-737 (1991)). For further details seedescription of “SUBTILASES” above. A parent subtilase may also be asubtilase isolated from a natural source, wherein subsequentmodification have been made while retaining the characteristic of asubtilase.

Alternatively the term “parent subtilase” may be termed “wild-typesubtilase”.

Modification(s) of a Subtilase Variant

The term “modification(s)” used in connection with modification(s) of asubtilase variant as discussed herein is defined to include chemicalmodification as well as genetic manipulation. The modification(s) can beby substitution, deletion and/or insertions in or at the amino acid(s)of interest.

Subtilase Variant

In the context of this invention, the term subtilase variant or mutatedsubtilase means a subtilase that has been produced by an organism whichis expressing a mutant gene derived from a parent microorganism whichpossessed an original or parent gene and which produced a correspondingparent enzyme, the parent gene having been mutated in order to producethe mutant gene from which said mutated subtilase protease is producedwhen expressed in a suitable host.

Homologous Subtilase Sequences

Specific amino acid residues of SAVINASE® are identified formodification herein to obtain a subtilase variant of the invention.

However, the invention is not limited to modifications of thisparticular subtilase, but extend to other parent (wild-type) subtilases,which have a homologous primary structure to that of SAVINASE®.

In order to identify other homologous subtilases, within the scope ofthis invention, an alignment of said subtilase(s) to a group ofpreviously aligned subtilases is performed keeping the previousalignment constant. A comparison to 18 highly conserved residues insubtilases is performed. The 18 highly conserved residues are shown inTable I (see Siezen et al. for further details relating to saidconserved residues).

TABLE I 18 highly conserved residues in subtilases Position: Conservedresidue 23 G 32 D 34 G 39 H 64 H 65 G 66 T 70 G 83 G 125 S 127 G 146 G154 G 155 N 219 G 220 T 221 S 225 P

After aligning allowing for necessary insertions and deletions in orderto maintain the alignment, suitable homologous residues are identified.Said homologous residues can then be modified according to theinvention.

Using the CLUSTALW (version 1.5, April 1995) computer alignment program(Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994) Nucleic AcidsResearch, 22:467-34680.), with GAP open penalty of 10.0 and GAPextension penalty of 0.1, using the BLOSUM30 protein weight matrix,alignment of a given subtilase to a group of previously alignedsubtilases is achieved using the Profile alignments option in theprogram. For a given subtilase to be within the scope of the invention,preferably 100% of the 18 highly conserved residues should be conserved.However, alignment of greater than or equal to 17 out of the 18residues, or as little as 16 of said conserved residues is also adequateto identify homologous residues. Conservation of the, in subtilases,catalytic triad Asp32/His64/Ser221 should be maintained.

The previously defined alignment is shown in FIGS. 1A and 1B, where thepercent identity of the individual subtilases in this alignment to the18 highly conserved residues also is shown.

Based on this description, it is routine for a person skilled in the artto identify suitable homologous subtilases and corresponding homologousresidues, which can be modified according to the invention. For example,Table II below shows a limited list of homologous subtilases andcorresponding suitable residues to be modified according to theinvention.

TABLE II Homologous Subtilases and corresponding homologous residuessuitable to be modified according to the invention. Enz. Pos BASBPNBYSYAB BLS309 BLS147 TVTHER 252 + 255 N252L + N252L + N252L + Q252L +N252L + T255I T255I T255I T255I D255I 252 + 255 N252V + N252V + N252V +Q252V + N252V + T255A T255A T255A T255A D255A 252 + 255 + N252M +N252M + N252M + Q252M + N252M + 259 T255C + T255C + T255C + T255C +D255C + D259H N259H S259H S259H G259H

It is obvious that a similar or larger table covering other homologoussubtilases may be easily produced by a person skilled in the art.

Wash Performance

The ability of an enzyme to catalyze the degradation of variousnaturally occurring substrates present on the objects to be cleanedduring e.g., wash is often referred to as its washing ability,washability, detergency, or wash performance. Throughout thisapplication the term wash performance will be used to encompass thisproperty.

Isolated DNA Sequences

The term “isolated”, when applied to a DNA sequence molecule, denotesthat the DNA sequence has been removed from its natural genetic milieuand is thus free of other extraneous or unwanted coding sequences, andis in a form suitable for use within genetically engineered proteinproduction systems. Such isolated molecules are those that are separatedfrom their natural environment and include cDNA and genomic clones.Isolated DNA molecules of the present invention are free of other geneswith which they are ordinarily associated, but may include naturallyoccurring 5′ and 3′ untranslated regions such as promoters andterminators. The identification of associated regions will be evident toone of ordinary skill in the art (see for example, Dynan and Tijan,Nature 316: 774-78 (1985)). The term “an isolated DNA sequence” mayalternatively be termed “a cloned DNA sequence”.

Isolated Proteins

When applied to a protein, the term “isolated” indicates that theprotein is found in a condition other than its native environment. In apreferred form, the isolated protein is substantially free of otherproteins, particularly other homologous proteins (i.e., “homologousimpurities” (see below)). It is preferred to provide the protein in ahighly purified form, i.e., greater than 40% pure, greater than 60%pure, greater than 80% pure, more preferably greater than 95% pure, andeven more preferably greater than 99% pure, as determined by SDS-PAGE.

The term “isolated protein” may alternatively be termed “purifiedprotein”.

Homologous Impurities

The term “homologous impurities” means any impurity (e.g., anotherpolypeptide than the polypeptide of the invention) which originate fromthe homologous cell where the polypeptide of the invention is originallyobtained from.

Obtained From

The term “obtained from” as used herein in connection with a specificmicrobial source, means that the polynucleotide and/or polypeptideproduced by the specific source, or by a cell in which a gene from thesource have been inserted.

Substrates

The term “Substrate” used in connection with a substrate for a proteaseis should be interpreted in its broadest form as comprising a compoundcontaining at least one peptide bond susceptible to hydrolysis by asubtilisin protease.

Products

The term “product” used in connection with a product derived from aprotease enzymatic reaction should in the context of this invention beinterpreted to include the products of a hydrolysis reaction involving asubtilase protease. A product may be the substrate in a subsequenthydrolysis reaction.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show an alignment of a number of homologous subtilases(SEQ ID NOS: 1-10), which are aligned to 18 highly conserved residues insubtilases. 18 highly conserved residues are highlighted in bold. Allshown subtilases, except JP170, have 100% identity in said conservedresidues. JP170 is a protease that has an “N” instead of “G” inconserved residue G146.

DETAILED DESCRIPTION OF THE INVENTION

Subtilase variants with improved wash performance:

The present inventors have identified variants of BLS309 (SAVINASE®)that have improved wash performance.

Accordingly, an embodiment of the invention relates to a subtilaseenzyme variant, wherein the modification is chosen from the groupcomprising:

N252L+T255I

N252V+T255A

N252M+T255C+S259H

N252S+T255E+S259C

N252K+T255S+S259C; or

a variant comprising one or more conservative modification(s) in any ofthe above mentioned variants (e.g., a conservative modification of N252L(hydrophobic amino acid)+T255I includes variants such as N252I(hydrophobic amino acid)+T255I, and N252V (hydrophobic aminoacid)+T255I.

Numerous subtilase variants of the invention were tested and showimproved wash performance in detergents (see the Examples below).

It is well known in the art that substitution of one amino acid to asimilar conservative amino acid only give a minor change in thecharacteristic of the enzyme.

Table III below list groups of conservative amino acids.

TABLE III Conservative amino acid substitutions Basic: arginine lysinehistidine Acidic: glutamic acid aspartic acid Polar: glutamineasparagine Hydrophobic: leucine isoleucine valine Aromatic:phenylalanine tryptophan tyrosine Small: glycine alanine serinethreonine methionine

Accordingly, subtilase variants such as 252L+255I, 252I+255I, and252V+255I will have a similar wash performance improvement. Further,subtilase variants such as N252L+T255I, N252I+T255I, and N252V+T255Iwill have a similar wash performance improvement.

Based on the disclosed subtilase variants herein, it is routine work,for a person skilled in the art, to identify further suitableconservative substitutions in order to obtain a subtilase variant withimproved wash performance.

In embodiments of the invention, the subtilases of interest are thosebelonging to the subgroups I-S1 and I-S2.

Relating to subgroup I-S1 preferred parent subtilase is ABSS168, BASBPN,BSSDY, BLSCAR or functional variants thereof having retained thecharacteristic of sub-group I-S1.

Relating to subgroup I-S2 preferred parent subtilase is BLS147, BLS309,BAPB92, TVTHER, BYSYAB or functional variants thereof having retainedthe characteristic of sub-group I-S2.

The present invention also comprises any one or more modifications inthe above mentioned positions in combination with any other modificationto the amino acid sequence of the parent enzyme. Especially combinationswith other modifications known in the art to provide improved propertiesto the enzyme are envisaged. The art describes a number of subtilasevariants with different improved properties and a number of those arementioned in the “Background of the invention” section above. Thosereferences are disclosed here as references to identify a subtilasevariant, which advantageously can be combined with a subtilase variantof the invention.

Such combinations comprise the positions: 222 (improve oxidationstability), 218 (improves thermal stability), substitutions in theCa-binding sites stabilising the enzyme, e.g., position 76, and manyother apparent from the prior art.

In further embodiments a subtilase variant of the invention mayadvantageously be combined with one or more modification(s) in any ofthe positions:

27, 36, 57, 76, 97, 101, 104, 120, 123, 167, 170, 206, 218, 222, 224,235 and 274.

Specifically the following BLS309 and BAPB92 variants are consideredappropriate for combination:

K27R, *36D, S57P, N76D, G97N, S101G, V104A, V104N, V104Y, H120D, N123S,Y167A, Y167I R170L, R170N, R170S, Q206E, N218S, M222A, M222S, T224S,K235L and T274A.

Furthermore variants comprising any of the variantsK27R+V104Y+N123S+T274A, N76D+V104A, V104N+S101G, or other combinationsof these mutations (K27R, N76D, S101G, V104A, V104N, V104Y, N123S,T274A), in combination with any one or more of the modification(s)mentioned above exhibit improved properties.

Even further subtilase variants of the main aspect(s) of the inventionare preferably combined with one or more modification(s) at any of thepositions 129, 131, 133 and 194, preferably as 129K, 131H, 133D, 133P,and 194P modifications, and most preferably as P129K, P131H, A133D,A133P, and A194P modifications. Any of those modification(s) may give ahigher expression level of a subtilase variant of the invention.

Methods for Producing Mutations in Subtilase Genes

Many methods for cloning a subtilase of the invention and forintroducing mutations into genes (e.g., subtilase genes) are well knownin the art.

In general standard procedures for cloning of genes and introducingmutations (random and/or site directed) into said genes may be used inorder to obtain a subtilase variant of the invention. For furtherdescription of suitable techniques reference is made to the Examplesbelow and Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Lab., Cold Spring Harbor, N.Y. (1989); Ausubel, F. M. etal. (eds.) “Current Protocols in Molecular Biology”. John Wiley andSons, 1995; Harwood, C. R., and Cutting, S. M. (eds.) “MolecularBiological Methods for Bacillus”. John Wiley and Sons, 1990); and WO96/34946.

Expression Vectors

A recombinant expression vector comprising a DNA construct encoding theenzyme of the invention may be any vector which may conveniently besubjected to recombinant DNA procedures, and the choice of vector willoften depend on the host cell into which it is to be introduced. Thus,the vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid. Alternatively,the vector may be one which, when introduced into a host cell, isintegrated into the host cell genome in part or in its entirety andreplicated together with the chromosome(s) into which it has beenintegrated.

The vector is preferably an expression vector in which the DNA sequenceencoding the enzyme of the invention is operably linked to additionalsegments required for transcription of the DNA. In general, theexpression vector is derived from plasmid or viral DNA, or may containelements of both. The term, “operably linked” indicates that thesegments are arranged so that they function in concert for theirintended purposes, e.g., transcription initiates in a promoter andproceeds through the DNA sequence coding for the enzyme.

The promoter may be any DNA sequence which shows transcriptionalactivity in the host cell of choice and may be derived from genesencoding proteins either homologous or heterologous to the host cell.

Examples of suitable promoters for use in bacterial host cells includethe promoter of the Bacillus stearothermophilus maltogenic amylase gene,the Bacillus licheniformis alpha-amylase gene, the Bacillusamyloliquefaciens alpha-amylase gene, the Bacillus subtilis alkalineprotease gene, or the Bacillus pumilus xylosidase gene, or the phageLambda P_(R) or P_(L) promoters or the E. coli lac, trp or tacpromoters.

The DNA sequence encoding the enzyme of the invention may also, ifnecessary, be operably connected to a suitable terminator.

The recombinant vector of the invention may further comprise a DNAsequence enabling the vector to replicate in the host cell in question.

The vector may also comprise a selectable marker, e.g., a gene theproduct of which complements a defect in the host cell, or a geneencoding resistance to e.g., antibiotics like kanamycin,chloramphenicol, erythromycin, tetracycline, spectinomycine, or thelike, or resistance to heavy metals or herbicides.

To direct an enzyme of the present invention into the secretory pathwayof the host cells, a secretory signal sequence (also known as a leadersequence, prepro sequence or pre sequence) may be provided in therecombinant vector. The secretory signal sequence is joined to the DNAsequence encoding the enzyme in the correct reading frame. Secretorysignal sequences are commonly positioned 5′ to the DNA sequence encodingthe enzyme. The secretory signal sequence may be that normallyassociated with the enzyme or may be from a gene encoding anothersecreted protein.

The procedures used to ligate the DNA sequences coding for the presentenzyme, the promoter and optionally the terminator and/or secretorysignal sequence, respectively, or to assemble these sequences bysuitable PCR amplification schemes, and to insert them into suitablevectors containing the information necessary for replication orintegration, are well known to persons skilled in the art (cf., forinstance, Sambrook et al., op. cit.).

Host Cells

The DNA sequence encoding the present enzyme introduced into the hostcell may be either homologous or heterologous to the host in question.If homologous to the host cell, i.e., produced by the host cell innature, it will typically be operably connected to another promotersequence or, if applicable, another secretory signal sequence and/orterminator sequence than in its natural environment. The term“homologous” is intended to include a DNA sequence encoding an enzymenative to the host organism in question. The term “heterologous” isintended to include a DNA sequence not expressed by the host cell innature. Thus, the DNA sequence may be from another organism, or it maybe a synthetic sequence.

The host cell into which the DNA construct or the recombinant vector ofthe invention is introduced may be any cell which is capable ofproducing the present enzyme and includes bacteria, yeast, fungi andhigher eukaryotic cells.

Examples of bacterial host cells which, on cultivation, are capable ofproducing the enzyme of the invention are gram-positive bacteria such asstrains of Bacillus, such as strains of B. alkalophilus, B.amyloliquefaciens, B. brevis, B. circulans, B. coagulans, B. lautus, B.lentus, B. licheniformis, B. megatherium, B. stearothermophilus, B.subtilis, or B. thuringiensis, or strains of Streptomyces, such as S.lividans or S. murinus, or gram-negative bacteria such as Echerichiacoli. The transformation of the bacteria may be effected by protoplasttransformation, electroporation, conjugation, or by using competentcells in a manner known per se (cf. Sambrook et al., supra).

When expressing the enzyme in bacteria such as E. coli, the enzyme maybe retained in the cytoplasm, typically as insoluble granules (known asinclusion bodies), or may be directed to the periplasmic space by abacterial secretion sequence. In the former case, the cells are lysedand the granules are recovered and denatured after which the enzyme isrefolded by diluting the denaturing agent. In the latter case, theenzyme may be recovered from the periplasmic space by disrupting thecells, e.g., by sonication or osmotic shock, to release the contents ofthe periplasmic space and recovering the enzyme.

When expressing the enzyme in gram-positive bacteria such as Bacillus orStreptomyces strains, the enzyme may be retained in the cytoplasm, ormay be directed to the extracellular medium by a bacterial secretionsequence. In the latter case, the enzyme may be recovered from themedium as described below.

Methods of Producing Subtilase

The present invention provides a method of producing an isolated enzymeaccording to the invention, wherein a suitable host cell, which has beentransformed with a DNA sequence encoding the enzyme, is cultured underconditions permitting the production of the enzyme, and the resultingenzyme is recovered from the culture.

When an expression vector comprising a DNA sequence encoding the enzymeis transformed into a heterologous host cell it is possible to enableheterologous recombinant production of the enzyme of the invention.

Thereby it is possible to make a highly purified subtilase composition,characterized in being free from homologous impurities.

In this context, homologous impurities means any impurities (e.g., otherpolypeptides than the enzyme of the invention) which originate from thehomologous cell where the enzyme of the invention is originally obtainedfrom.

The medium used to culture the transformed host cells may be anyconventional medium suitable for growing the host cells in question. Theexpressed subtilase may conveniently be secreted into the culture mediumand may be recovered therefrom by well-known procedures includingseparating the cells from the medium by centrifugation or filtration,precipitating proteinaceous components of the medium by means of a saltsuch as ammonium sulphate, followed by chromatographic procedures suchas ion exchange chromatography, affinity chromatography, or the like.

Use of a Subtilase Variant of the Invention

A subtilase protease variant of the invention may be used for a numberof industrial applications, in particular within the detergent industry.

Further the invention relates to an enzyme composition, which comprisesa subtilase variant of the invention.

A summary of preferred industrial applications and correspondingpreferred enzyme compositions are described below.

This summary is not in any way intended to be a complete list ofsuitable applications of a subtilase variant of the invention. Asubtilase variant of the invention may be used in other industrialapplications known in the art to include use of a protease, inparticular a subtilase.

Detergent Compositions Comprising the Mutant Enzymes

The present invention comprises the use of the mutant enzymes of theinvention in cleaning and detergent compositions and such compositionscomprising the mutant subtilisin enzymes. Such cleaning and detergentcompositions are well described in the art and reference is made to WO96/34946, WO 97/07202, and WO 95/30011 for further description ofsuitable cleaning and detergent compositions.

Further reference is made to the Examples below showing wash performanceimprovements for a number of subtilase variants of the invention.

Detergent Disclosure and Examples

Surfactant System

The detergent compositions according to the present invention comprise asurfactant system, wherein the surfactant can be selected from nonionicand/or anionic and/or cationic and/or ampholytic and/or zwitterionicand/or semi-polar surfactants.

The surfactant is typically present at a level from 0.1-60% by weight.

The surfactant is preferably formulated to be compatible with enzymecomponents present in the composition. In liquid or gel compositions thesurfactant is most preferably formulated in such a way that it promotes,or at least does not degrade, the stability of any enzyme in thesecompositions.

Preferred systems to be used according to the present invention compriseas a surfactant one or more of the nonionic and/or anionic surfactantsdescribed herein.

Polyethylene, polypropylene, and polybutylene oxide condensates of alkylphenols are suitable for use as the nonionic surfactant of thesurfactant systems of the present invention, with the polyethylene oxidecondensates being preferred. These compounds include the condensationproducts of alkyl phenols having an alkyl group containing from about 6to about 14 carbon atoms, preferably from about 8 to about 14 carbonatoms, in either a straight chain or branched-chain configuration withthe alkylene oxide. In a preferred embodiment, the ethylene oxide ispresent in an amount equal to from about 2 to about 25 moles, morepreferably from about 3 to about 15 moles, of ethylene oxide per mole ofalkyl phenol. Commercially available nonionic surfactants of his typeinclude Igepal™ CO-630, marketed by the GAF Corporation; and Triton™X-45, X-114, X-100 and X-102, all marketed by the Rohm & Haas Company.These surfactants are commonly referred to as alkylphenol alkoxylates(e.g., alkyl phenol ethoxylates).

The condensation products of primary and secondary aliphatic alcoholswith about 1 to about 25 moles of ethylene oxide are suitable for use asthe nonionic surfactant of the nonionic surfactant systems of thepresent invention. The alkyl chain of the aliphatic alcohol can eitherbe straight or branched, primary or secondary, and generally containsfrom about 8 to about 22 carbon atoms. Preferred are the condensationproducts of alcohols having an alkyl group containing from about 8 toabout 20 carbon atoms, more preferably from about 10 to about 18 carbonatoms, with from about 2 to about 10 moles of ethylene oxide per mole ofalcohol. About 2 to about 7 moles of ethylene oxide and most preferablyfrom 2 to 5 moles of ethylene oxide per mole of alcohol are present insaid condensation products. Examples of commercially available nonionicsurfactants of this type include Tergitol™ 15-S-9 (The condensationproduct of C₁₁-C₁₅ linear alcohol with 9 moles ethylene oxide),Tergitol™ 24-L-6 NMW (the condensation product of C₁₂-C₁₄ primaryalcohol with 6 moles ethylene oxide with a narrow molecular weightdistribution), both marketed by Union Carbide Corporation; Neodol™ 45-9(the condensation product of C₁₄-C₁₅ linear alcohol with 9 moles ofethylene oxide), Neodol™ 23-3 (the condensation product of C₁₂-C₁₃linear alcohol with 3.0 moles of ethylene oxide), Neodol™ 45-7 (thecondensation product of C₁₄-C₁₅ linear alcohol with 7 moles of ethyleneoxide), Neodol™ 45-5 (the condensation product of C₁₄-C₁₅ linear alcoholwith 5 moles of ethylene oxide) marketed by Shell Chemical Company,Kyro™ EOB (the condensation product of C₁₃-C₁₅ alcohol with 9 molesethylene oxide), marketed by The Procter & Gamble Company, and GenapolLA 050 (the condensation product of C₁₂-C₁₄ alcohol with 5 moles ofethylene oxide) marketed by Hoechst. Preferred range of HLB in theseproducts is from 8-11 and most preferred from 8-10.

Also useful as the nonionic surfactant of the surfactant systems of thepresent invention are alkylpolysaccharides disclosed in U.S. Pat. No.4,565,647, having a hydrophobic group containing from about 6 to about30 carbon atoms, preferably from about 10 to about 16 carbon atoms and apolysaccharide, e.g., a polyglycoside, hydrophilic group containing fromabout 1.3 to about 10, preferably from about 1.3 to about 3, mostpreferably from about 1.3 to about 2.7 saccharide units. Any reducingsaccharide containing 5 or 6 carbon atoms can be used, e.g., glucose,galactose and galactosyl moieties can be substituted for the glucosylmoieties (optionally the hydrophobic group is attached at the 2-, 3-,4-, etc. positions thus giving a glucose or galactose as opposed to aglucoside or galactoside). The intersaccharide bonds can be, e.g.,between the one position of the additional saccharide units and the 2-,3-, 4-, and/or 6-positions on the preceding saccharide units.

The preferred alkylpolyglycosides have the formulaR²O(C_(n)H_(2n)O)_(t)(glycosyl)_(x)wherein R² is selected from the group consisting of alkyl, alkylphenyl,hydroxyalkyl, hydroxyalkylphenyl, and mixtures thereof in which thealkyl groups contain from about 10 to about 18, preferably from about 12to about 14, carbon atoms; n is 2 or 3, preferably 2; t is from 0 toabout 10, preferably 0; and x is from about 1.3 to about 10, preferablyfrom about 1.3 to about 3, most preferably from about 1.3 to about 2.7.The glycosyl is preferably derived from glucose. To prepare thesecompounds, the alcohol or alkylpolyethoxy alcohol is formed first andthen reacted with glucose, or a source of glucose, to form the glucoside(attachment at the 1-position). The additional glycosyl units can thenbe attached between their 1-position and the preceding glycosyl units2-, 3-, 4-, and/or 6-position, preferably predominantly the 2-position.

The condensation products of ethylene oxide with a hydrophobic baseformed by the condensation of propylene oxide with propylene glycol arealso suitable for use as the additional nonionic surfactant systems ofthe present invention. The hydrophobic portion of these compounds willpreferably have a molecular weight from about 1500 to about 1800 andwill exhibit water insolubility. The addition of polyoxyethylenemoieties to this hydrophobic portion tends to increase the watersolubility of the molecule as a whole, and the liquid character of theproduct is retained up to the point where the polyoxyethylene content isabout 50% of the total weight of the condensation product, whichcorresponds to condensation with up to about 40 moles of ethylene oxide.Examples of compounds of this type include certain of the commerciallyavailable Pluronic™ surfactants, marketed by BASF.

Also suitable for use as the nonionic surfactant of the nonionicsurfactant system of the present invention, are the condensationproducts of ethylene oxide with the product resulting from the reactionof propylene oxide and ethylenediamine. The hydrophobic moiety of theseproducts consists of the reaction product of ethylenediamine and excesspropylene oxide, and generally has a molecular weight of from about 2500to about 3000. This hydrophobic moiety is condensed with ethylene oxideto the extent that the condensation product contains from about 40% toabout 80% by weight of polyoxyethylene and has a molecular weight offrom about 5,000 to about 11,000. Examples of this type of nonionicsurfactant include certain of the commercially available Tetronic™compounds, marketed by BASF.

Preferred for use as the nonionic surfactant of the surfactant systemsof the present invention are polyethylene oxide condensates of alkylphenols, condensation products of primary and secondary aliphaticalcohols with from about 1 to about 25 moles of ethyleneoxide,alkylpolysaccharides, and mixtures hereof. Most preferred are C₈-C₁₄alkyl phenol ethoxylates having from 3 to 15 ethoxy groups and C₈-C₁₈alcohol ethoxylates (preferably C₁₀ avg.) having from 2 to 10 ethoxygroups, and mixtures thereof.

Highly preferred nonionic surfactants are polyhydroxy fatty acid amidesurfactants of the formula

wherein R¹ is H, or R¹ is C₁₋₄ hydrocarbyl, 2-hydroxyethyl,2-hydroxypropyl or a mixture thereof, R² is C₅₋₃₁ hydrocarbyl, and Z isa polyhydroxyhydrocarbyl having a linear hydrocarbyl chain with at least3 hydroxyls directly connected to the chain, or an alkoxylatedderivative thereof. Preferably, R¹ is methyl, R² is straight C₁₁₋₁₅alkyl or C₁₋₁₈ alkyl or alkenyl chain such as coconut alkyl or mixturesthereof, and Z is derived from a reducing sugar such as glucose,fructose, maltose or lactose, in a reductive amination reaction.

Highly preferred anionic surfactants include alkyl alkoxylated sulfatesurfactants. Examples hereof are water soluble salts or acids of theformula RO(A)_(m)SO₃M wherein R is an unsubstituted C₁₀-C-₂₄ alkyl orhydroxyalkyl group having a C₁₀-C₂₄ alkyl component, preferably aC₁₂-C₂₀ alkyl or hydroxyalkyl, more preferably C₁₂-C₁₈ alkyl orhydroxyalkyl, A is an ethoxy or propoxy unit, m is greater than zero,typically between about 0.5 and about 6, more preferably between about0.5 and about 3, and M is H or a cation which can be, for example, ametal cation (e.g., sodium, potassium, lithium, calcium, magnesium,etc.), ammonium or substituted-ammonium cation. Alkyl ethoxylatedsulfates as well as alkyl propoxylated sulfates are contemplated herein.Specific examples of substituted ammonium cations include methyl,dimethyl, trimethyl-ammonium cations and quaternary ammonium cationssuch as tetramethyl-ammonium and dimethyl piperdinium cations and thosederived from alkylamines such as ethylamine, diethylamine,triethylamine, mixtures thereof, and the like. Exemplary surfactants areC₁₂-C₁₈ alkyl polyethoxylate (1.0) sulfate (C₁₂-C₁₈E(1.0)M), C₁₂-C₁₈alkyl polyethoxylate (2.25) sulfate (C₁₂-C₁₈(2.25)M, and C₁₂-C₁₈ alkylpolyethoxylate (3.0) sulfate (C₁₂-C₁₈E(3.0)M), and C₁₂-C₁₈ alkylpolyethoxylate (4.0) sulfate (C₁₂-C₁₈E(4.0)M), wherein M is selectedfrom sodium and potassium.

Suitable anionic surfactants to be used are alkyl ester sulfonatesurfactants including linear esters of C₈-C₂₀ carboxylic acids (i.e.,fatty acids) which are sulfonated with gaseous SO₃ according to “TheJournal of the American Oil Chemists Society”, 52: 323-329 (1975).Suitable starting materials would include natural fatty substances asderived from tallow, palm oil, etc.

The preferred alkyl ester sulfonate surfactant, especially for laundryapplications, comprise alkyl ester sulfonate surfactants of the formula:

wherein R³ is a C₈-C₂₀ hydrocarbyl, preferably an alkyl, or combinationthereof, R⁴ is a C₁-C₆ hydrocarbyl, preferably an alkyl, or combinationthereof, and M is a cation which forms a water soluble salt with thealkyl ester sulfonate. Suitable salt-forming cations include metals suchas sodium, potassium, and lithium, and substituted or unsubstitutedammonium cations, such as monoethanolamine, diethonolamine, andtriethanolamine. Preferably, R³ is C₁₀-C₁₆ alkyl, and R⁴ is methyl,ethyl or isopropyl. Especially preferred are the methyl ester sulfonateswherein R³ is C₁₀-C₁₆ alkyl.

Other suitable anionic surfactants include the alkyl sulfate surfactantswhich are water soluble salts or acids of the formula ROSO₃M wherein Rpreferably is a C₁₀-C₂₄ hydrocarbyl, preferably an alkyl or hydroxyalkylhaving a C₁₀-C₂₀ alkyl component, more preferably a C₁₂-C₁₈ alkyl orhydroxyalkyl, and M is H or a cation, e.g., an alkali metal cation(e.g., sodium, potassium, lithium), or ammonium or substituted ammonium(e.g., methyl, dimethyl, and trimethyl ammonium cations and quaternaryammonium cations such as tetramethyl ammonium and dimethyl piperdiniumcations and quaternary ammonium cations derived from alkylamines such asethylamine, diethylamine, triethylamine, and mixtures thereof, and thelike). Typically, alkyl chains of C₁₂-C₁₆ are preferred for lower washtemperatures (e.g., below about 50° C.) and C₁₆-C₁₈ alkyl chains arepreferred for higher wash temperatures (e.g., above about 50° C.).

Other anionic surfactants useful for detersive purposes can be includedin the laundry detergent compositions of the present invention. Thesesinclude salts (including, for example, sodium, potassium, ammonium, andsubstituted ammonium salts such as mono-, di- and triethanolamine salts)of soap, C₈-C₂₂ primary or secondary alkanesulfonates, C₈-C₂₄olefinsulfonates, sulfonated polycarboxylic acids prepared bysulfonation of the pyrolyzed product of alkaline earth metal citrates,e.g., as described in British patent specification no. 1,082,179, C₈-C₂₄alkylpolyglycolethersulfates (containing up to 10 moles of ethyleneoxide); alkyl glycerol sulfonates, fatty acyl glycerol sulfonates, fattyoleyl glycerol sulfates, alkyl phenol ethylene oxide ether sulfates,paraffin sulfonates, alkyl phosphates, isethionates such as the acylisethionates, N-acyl taurates, alkyl succinamates and sulfosuccinates,monoesters of sulfosuccinates (especially saturated and unsaturatedC₁₂-C₁₈ monoesters) and diesters of sulfosuccinates (especiallysaturated and unsaturated C₆-C₁₂ diesters), acyl sarcosinates, sulfatesof alkylpolysaccharides such as the sulfates of alkylpolyglucoside (thenonionic nonsulfated compounds being described below), branched primaryalkyl sulfates, and alkyl polyethoxy carboxylates such as those of theformula RO(CH₂CH₂O)_(k)—CH₂COO-M+ wherein R is a C₈-C₂₂ alkyl, k is aninteger from 1 to 10, and M is a soluble salt forming cation. Resinacids and hydrogenated resin acids are also suitable, such as rosin,hydrogenated rosin, and resin acids and hydrogenated resin acids presentin or derived from tall oil.

Alkylbenzene sulfonates are highly preferred. Especially preferred arelinear (straight-chain) alkyl benzene sulfonates (LAS) wherein the alkylgroup preferably contains from 10 to 18 carbon atoms.

Further examples are described in “Surface Active Agents and Detergents”(Vol. I and II by Schwartz, Perrry and Berch). A variety of suchsurfactants are also generally disclosed in U.S. Pat. No. 3,929,678(column 23, line 58 through column 29, line 23, which is incorporatedherein by reference).

When included therein, the laundry detergent compositions of the presentinvention typically comprise from about 1% to about 40%, preferably fromabout 3% to about 20% by weight of such anionic surfactants.

The laundry detergent compositions of the present invention may alsocontain cationic, ampholytic, zwitterionic, and semi-polar surfactants,as well as the nonionic and/or anionic surfactants other than thosealready described herein.

Cationic detersive surfactants suitable for use in the laundry detergentcompositions of the present invention are those having one long-chainhydrocarbyl group. Examples of such cationic surfactants include theammonium surfactants such as alkyltrimethylammonium halogenides, andhose surfactants having the formula:[R²(OR³)_(y)][R⁴(OR³)_(y)]₂R⁵N+X−wherein R² is an alkyl or alkyl benzyl group having from about 8 toabout 18 carbon atoms in the alkyl chain, each R³ is selected form thegroup consisting of —CH₂CH₂—, —CH₂CH(CH₃)—, —CH₂CH(CH₂OH)—, —CH₂CH₂CH₂—,and mixtures thereof; each R⁴ is selected from the group consisting ofC₁-C₄ alkyl, C₁-C₄ hydroxyalkyl, benzyl ring structures formed byjoining the two R⁴ groups, —CH₂CHOHCHOHCOR⁶CHOHCH₂OH, wherein R⁶ is anyhexose or hexose polymer having a molecular weight less than about 1000,and hydrogen when y is not 0; R⁵ is the same as R⁴ or is an alkyl chain,wherein the total number of carbon atoms or R² plus R⁵ is not more thanabout 18; each y is from 0 to about 10, and the sum of the y values isfrom 0 to about 15; and X is any compatible anion.

Highly preferred cationic surfactants are the water soluble quaternaryammonium compounds useful in the present composition having the formula:R₁R₂R₃R₄N⁺X⁻  (i)wherein R₁ is C₈-C₁₆ alkyl, each of R₂, R₃ and R₄ is independently C₁-C₄alkyl, C₁-C₄ hydroxy alkyl, benzyl, and —(C₂H₄₀)_(x)H where x has avalue from 2 to 5, and X is an anion. Not more than one of R₂, R₃ or R₄should be benzyl.

The preferred alkyl chain length for R₁ is C₁₂-C₁₅, particularly wherethe alkyl group is a mixture of chain lengths derived from coconut orpalm kernel fat or is derived synthetically by olefin build up or OXOalcohols synthesis.

Preferred groups for R₂, R₃ and R4 are methyl and hydroxyethyl groupsand the anion X may be selected from halide, methosulphate, acetate andphosphate ions.

Examples of suitable quaternary ammonium compounds of formula (i) foruse herein are:

-   -   coconut trimethyl ammonium chloride or bromide;    -   coconut methyl dihydroxyethyl ammonium chloride or bromide;    -   decyl triethyl ammonium chloride;    -   decyl dimethyl hydroxyethyl ammonium chloride or bromide;    -   C₁₂₋₁₅ dimethyl hydroxyethyl ammonium chloride or bromide;    -   coconut dimethyl hydroxyethyl ammonium chloride or bromide;    -   myristyl trimethyl ammonium methyl sulphate;    -   lauryl dimethyl benzyl ammonium chloride or bromide;    -   lauryl dimethyl (ethenoxy)₄ ammonium chloride or bromide;    -   choline esters (compounds of formula (i) wherein R₁ is

-   -   di-alkyl imidazolines [compounds of formula (i)].

Other cationic surfactants useful herein are also described in U.S. Pat.No. 4,228,044 and in EP 000 224.

When included therein, the laundry detergent compositions of the presentinvention typically comprise from 0.2% to about 25%, preferably fromabout 1% to about 8% by weight of such cationic surfactants.

Ampholytic surfactants are also suitable for use in the laundrydetergent compositions of the present invention. These surfactants canbe broadly described as aliphatic derivatives of secondary or tertiaryamines, or aliphatic derivatives of heterocyclic secondary and tertiaryamines in which the aliphatic radical can be straight- orbranched-chain. One of the aliphatic substituents contains at leastabout 8 carbon atoms, typically from about 8 to about 18 carbon atoms,and at least one contains an anionic water-solubilizing group, e.g.,carboxy, sulfonate, sulfate. See U.S. Pat. No. 3,929,678 (column 19,lines 18-35) for examples of ampholytic surfactants.

When included therein, the laundry detergent compositions of the presentinvention typically comprise from 0.2% to about 15%, preferably fromabout 1% to about 10% by weight of such ampholytic surfactants.

Zwitterionic surfactants are also suitable for use in laundry detergentcompositions. These surfactants can be broadly described as derivativesof secondary and tertiary amines, derivatives of heterocyclic secondaryand tertiary amines, or derivatives of quaternary ammonium, quaternaryphosphonium or tertiary sulfonium compounds. See U.S. Pat. No. 3,929,678(column 19, line 38 through column 22, line 48) for examples ofzwitterionic surfactants.

When included therein, the laundry detergent compositions of the presentinvention typically comprise from 0.2% to about 15%, preferably fromabout 1% to about 10% by weight of such zwitterionic surfactants.

Semi-polar nonionic surfactants are a special category of nonionicsurfactants which include water-soluble amine oxides containing onealkyl moiety of from about 10 to about 18 carbon atoms and 2 moietiesselected from the group consisting of alkyl groups and hydroxyalkylgroups containing from about 1 to about 3 carbon atoms; water solublephosphine oxides containing one alkyl moiety of from about 10 to about18 carbon atoms and 2 moieties selected from the group consisting ofalkyl groups and hydroxyalkyl groups containing from about 1 to about 3carbon atoms; and water-soluble sulfoxides containing one alkyl moietyfrom about 10 to about 18 carbon atoms and a moiety selected from thegroup consisting of alkyl and hydroxyalkyl moieties of from about 1 toabout 3 carbon atoms.

Semi-polar nonionic detergent surfactants include the amine oxidesurfactants having the formula:

wherein R³ is an alkyl, hydroxyalkyl, or alkyl phenyl group or mixturesthereof containing from about 8 to about 22 carbon atoms; R⁴ is analkylene or hydroxyalkylene group containing from about 2 to about 3carbon atoms or mixtures thereof; x is from 0 to about 3: and each R⁵ isan alkyl or hydroxyalkyl group containing from about 1 to about 3 carbonatoms or a polyethylene oxide group containing from about 1 to about 3ethylene oxide groups. The R⁵ groups can be attached to each other,e.g., through an oxygen or nitrogen atom, to form a ring structure.

These amine oxide surfactants in particular include C₁₀-C₁₈ alkyldimethyl amine oxides and C₈-C₁₂ alkoxy ethyl dihydroxy ethyl amineoxides.

When included therein, the laundry detergent compositions of the presentinvention typically comprise from 0.2% to about 15%, preferably fromabout 1% to about 10% by weight of such semi-polar nonionic surfactants.

Builder system

The compositions according to the present invention may further comprisea builder system. Any conventional builder system is suitable for useherein including aluminosilicate materials, silicates, polycarboxylatesand fatty acids, materials such as ethylenediamine tetraacetate, metalion sequestrants such as aminopolyphosphonates, particularlyethylenediamine tetramethylene phosphonic acid and diethylene triaminepentamethylenephosphonic acid. Though less preferred for obviousenvironmental reasons, phosphate builders can also be used herein.

Suitable builders can be an inorganic ion exchange material, commonly aninorganic hydrated aluminosilicate material, more particularly ahydrated synthetic zeolite such as hydrated zeolite A, X, B, HS or MAP.

Another suitable inorganic builder material is layered silicate, e.g.,SKS-6 (Hoechst). SKS-6 is a crystalline layered silicate consisting ofsodium silicate (Na₂Si₂O₅).

Suitable polycarboxylates containing one carboxy group include lacticacid, glycolic acid and ether derivatives thereof as disclosed inBelgian Patent Nos. 831,368, 821,369 and 821,370. Polycarboxylatescontaining two carboxy groups include the water-soluble salts ofsuccinic acid, malonic acid, (ethylenedioxy) diacetic acid, maleic acid,diglycollic acid, tartaric acid, tartronic acid and fumaric acid, aswell as the ether carboxylates described in German Offenle-enschrift2,446,686, and 2,446,487, U.S. Pat. No. 3,935,257 and the sulfinylcarboxylates described in Belgian Patent No. 840,623. Polycarboxylatescontaining three carboxy groups include, in particular, water solublecitrates, aconitrates and citraconates as well as succinate derivativessuch as the carboxymethyloxysuccinates described in British Patent No.1,379,241, lactoxysuccinates described in Netherlands Application7205873, and the oxypolycarboxylate materials such as2-oxa-1,1,3-propane tricarboxylates described in British Patent No.1,387,447.

Polycarboxylates containing four carboxy groups include oxydisuccinatesdisclosed in British Patent No. 1,261,829, 1,1,2,2-ethanetetracarboxylates, 1,1,3,3-propane tetracarboxylates containing sulfosubstituents include the sulfosuccinate derivatives disclosed in BritishPatent Nos. 1,398,421 and 1,398,422 and in U.S. Pat. No. 3,936,448, andthe sulfonated pyrolysed citrates described in British Patent No.1,082,179, while polycarboxylates containing phosphone substituents aredisclosed in British Patent No. 1,439,000.

Alicyclic and heterocyclic polycarboxylates includecyclopentane-cis,cis-cis-tetracarboxylates, cyclopentadienidepentacarboxylates,2,3,4,5-tetrahydro-furan-cis,cis,cis-tetracarboxylates,2,5-tetrahydro-furan-cis-discarboxylates,2,2,5,5-tetrahydrofuran-tetracarboxylates,1,2,3,4,5,6-hexane-hexacarboxylates and carboxymethyl derivatives ofpolyhydric alcohols such as sorbitol, mannitol and xylitol. Aromaticpolycarboxylates include mellitic acid, pyromellitic acid and thephthalic acid derivatives disclosed in British Patent No. 1,425,343.

Of the above, the preferred polycarboxylates are hydroxy-carboxylatescontaining up to three carboxy groups per molecule, more particularlycitrates.

Preferred builder systems for use in the present compositions include amixture of a water-insoluble aluminosilicate builder such as zeolite Aor of a layered silicate (SKS-6), and a water soluble carboxylatechelating agent such as citric acid.

A suitable chelant for inclusion in the detergent compositions inaccordance with the invention is ethylenediamine-N,N′-disuccinic acid(EDDS) or the alkali metal, alkaline earth metal, ammonium, orsubstituted ammonium salts thereof, or mixtures thereof. Preferred EDDScompounds are the free acid form and the sodium or magnesium saltthereof. Examples of such preferred sodium salts of EDDS include Na₂EDDSand Na₄EDDS. Examples of such preferred magnesium salts of EDDS includeMgEDDS and Mg₂EDDS. The magnesium salts are the most preferred forinclusion in compositions in accordance with the invention.

Preferred builder systems include a mixture of a water-insolublealuminosilicate builder such as zeolite A, and a water solublecarboxylate chelating agent such as citric acid.

Other builder materials that can form part of the builder system for usein granular compositions include inorganic materials such as alkalimetal carbonates, bicarbonates, silicates, and organic materials such asthe organic phosphonates, amino polyalkylene phosphonates and aminopolycarboxylates.

Other suitable water-soluble organic salts are the homo- or co-polymericacids or their salts, in which the polycarboxylic acid comprises atleast two carboxyl radicals separated form each other by not more thantwo carbon atoms.

Polymers of this type are disclosed in GB-A-1,596,756. Examples of suchsalts are polyacrylates of MW 2000-5000 and their copolymers with maleicanhydride, such copolymers having a molecular weight of from 20,000 to70,000, especially about 40,000.

Detergency builder salts are normally included in amounts of from 5% to80% by weight of the composition. Preferred levels of builder for liquiddetergents are from 5% to 30%.

Enzymes

Preferred detergent compositions, in addition to the enzyme preparationof the invention, comprise other enzyme(s) which provides cleaningperformance and/or fabric care benefits.

Such enzymes include other proteases, lipases, cutinases, amylases,cellulases, peroxidases, oxidases (e.g., laccases).

Proteases: Any other protease suitable for use in alkaline solutions canbe used. Suitable proteases include those of animal, vegetable ormicrobial origin. Microbial origin is preferred. Chemically orgenetically modified mutants are included. The protease may be a serineprotease, preferably an alkaline microbial protease or a trypsin-likeprotease. Examples of alkaline proteases are subtilisins, especiallythose derived from Bacillus, e.g., subtilisin Novo, subtilisinCarlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (describedin WO 89/06279). Examples of trypsin-like proteases are trypsin (e.g.,of porcine or bovine origin) and the Fusarium protease described in WO89/06270.

Preferred commercially available protease enzymes include those soldunder the trade names Alcalase, Savinase, Primase, Durazym, and Esperaseby Novo Nordisk A/S (Denmark), those sold under the tradename Maxatase,Maxacal, Maxapem, Properase, Purafect and Purafect OXP by GenencorInternational, and those sold under the tradename Opticlean and Optimaseby Solvay Enzymes. Protease enzymes may be incorporated into thecompositions in accordance with the invention at a level of from0.00001% to 2% of enzyme protein by weight of the composition,preferably at a level of from 0.0001% to 1% of enzyme protein by weightof the composition, more preferably at a level of from 0.001% to 0.5% ofenzyme protein by weight of the composition, even more preferably at alevel of from 0.01% to 0.2% of enzyme protein by weight of thecomposition.

Lipases: Any lipase suitable for use in alkaline solutions can be used.Suitable lipases include those of bacterial or fungal origin. Chemicallyor genetically modified mutants are included.

Examples of useful lipases include a Humicola lanuginosa lipase, e.g.,as described in EP 258 068 and EP 305 216, a Rhizomucor miehei lipase,e.g., as described in EP 238 023, a Candida lipase, such as a C.antarctica lipase, e.g., the C. antarctica lipase A or B described in EP214 761, a Pseudomonas lipase such as a P. alcaligenes and P.pseudoalcaligenes lipase, e.g., as described in EP 218 272, a P. cepacialipase, e.g., as described in EP 331 376, a P. stutzeri lipase, e.g., asdisclosed in GB 1,372,034, a P. fluorescens lipase, a Bacillus lipase,e.g., a B. subtilis lipase (Dartois et al., Biochemica et Biophysicaacta 1131: 253-260 (1993)), a B. stearothermophilus lipase (JP64/744992) and a B. pumilus lipase (WO 91/16422).

Furthermore, a number of cloned lipases may be useful, including thePenicillium camembertii lipase described by Yamaguchi et al., Gene 103:61-67 (1991)), the Geotricum candidum lipase (Schimada, Y. et al., J.Biochem., 106: 383-388 (1989)), and various Rhizopus lipases such as aR. delemar lipase (Hass, M. J. et al., Gene 109: 117-113 (1991)), a R.niveus lipase (Kugimiya et al., Biosci. Biotech. Biochem. 56: 716-719(1992)) and a R. oryzae lipase.

Other types of lipolytic enzymes such as cutinases may also be useful,e.g., a cutinase derived from Pseudomonas mendocina as described in WO88/09367, or a cutinase derived from Fusarium solani pisi (e.g.,described in WO 90/09446).

Especially suitable lipases are lipases such as M1 Lipase™, Luma fast™and Lipomax™ (Genencor), Lipolase™ and Lipolase Ultra™ (Novo NordiskA/S), and Lipase P “Amano” (Amano Pharmaceutical Co. Ltd.).

The lipases are normally incorporated in the detergent composition at alevel of from 0.00001% to 2% of enzyme protein by weight of thecomposition, preferably at a level of from 0.0001% to 1% of enzymeprotein by weight of the composition, more preferably at a level of from0.001% to 0.5% of enzyme protein by weight of the composition, even morepreferably at a level of from 0.01% to 0.2% of enzyme protein by weightof the composition.

Amylases: Any amylase (alpha and/or beta) suitable for use in alkalinesolutions can be used. Suitable amylases include those of bacterial orfungal origin. Chemically or genetically modified mutants are included.Amylases include, for example, α-amylases obtained from a special strainof B. licheniformis, described in more detail in GB 1,296,839.Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™ andBAN™ (available from Novo Nordisk A/S) and Rapidase™ and Maxamyl P™(available from Genencor).

The amylases are normally incorporated in the detergent composition at alevel of from 0.00001% to 2% of enzyme protein by weight of thecomposition, preferably at a level of from 0.0001% to 1% of enzymeprotein by weight of the composition, more preferably at a level of from0.001% to 0.5% of enzyme protein by weight of the composition, even morepreferably at a level of from 0.01% to 0.2% of enzyme protein by weightof the composition.

Cellulases: Any cellulase suitable for use in alkaline solutions can beused. Suitable cellulases include those of bacterial or fungal origin.Chemically or genetically modified mutants are included. Suitablecellulases are disclosed in U.S. Pat. No. 4,435,307, which disclosesfungal cellulases produced from Humicola insolens. Especially suitablecellulases are the cellulases having color care benefits. Examples ofsuch cellulases are cellulases described in European patent applicationNo. 0 495 257.

Commercially available cellulases include Celluzyme™ produced by astrain of Humicola insolens, (Novo Nordisk A/S), and KAC-500(B)T (KaoCorporation).

Cellulases are normally incorporated in the detergent composition at alevel of from 0.00001% to 2% of enzyme protein by weight of thecomposition, preferably at a level of from 0.0001% to 1% of enzymeprotein by weight of the composition, more preferably at a level of from0.001% to 0.5% of enzyme protein by weight of the composition, even morepreferably at a level of from 0.01% to 0.2% of enzyme protein by weightof the composition.

Peroxidases/Oxidases: Peroxidase enzymes are used in combination withhydrogen peroxide or a source thereof (e.g., a percarbonate, perborateor persulfate). Oxidase enzymes are used in combination with oxygen.Both types of enzymes are used for “solution bleaching”, i.e., toprevent transfer of a textile dye from a dyed fabric to another fabricwhen said fabrics are washed together in a wash liquor, preferablytogether with an enhancing agent as described in e.g., WO 94/12621 andWO 95/01426. Suitable peroxidases/oxidases include those of plant,bacterial or fungal origin. Chemically or genetically modified mutantsare included.

Peroxidase and/or oxidase enzymes are normally incorporated in thedetergent composition at a level of from 0.00001% to 2% of enzymeprotein by weight of the composition, preferably at a level of from0.0001% to 1% of enzyme protein by weight of the composition, morepreferably at a level of from 0.001% to 0.5% of enzyme protein by weightof the composition, even more preferably at a level of from 0.01% to0.2% of enzyme protein by weight of the composition.

Mixtures of the above mentioned enzymes are encompassed herein, inparticular a mixture of a protease, an amylase, a lipase and/or acellulase.

The enzyme of the invention, or any other enzyme incorporated in thedetergent composition, is normally incorporated in the detergentcomposition at a level from 0.00001% to 2% of enzyme protein by weightof the composition, preferably at a level from 0.0001% to 1% of enzymeprotein by weight of the composition, more preferably at a level from0.001% to 0.5% of enzyme protein by weight of the composition, even morepreferably at a level from 0.01% to 0.2% of enzyme protein by weight ofthe composition.

Bleaching agents: Additional optional detergent ingredients that can beincluded in the detergent compositions of the present invention includebleaching agents such as PB1, PB4 and percarbonate with a particle sizeof 400-800 microns. These bleaching agent components can include one ormore oxygen bleaching agents and, depending upon the bleaching agentchosen, one or more bleach activators. When present oxygen bleachingcompounds will typically be present at levels of from about 1% to about25%. In general, bleaching compounds are optional added components innon-liquid formulations, e.g., granular detergents.

The bleaching agent component for use herein can be any of the bleachingagents useful for detergent compositions including oxygen bleaches aswell as others known in the art.

The bleaching agent suitable for the present invention can be anactivated or non-activated bleaching agent.

One category of oxygen bleaching agent that can be used encompassespercarboxylic acid bleaching agents and salts thereof. Suitable examplesof this class of agents include magnesium monoperoxyphthalatehexahydrate, the magnesium salt of meta-chloro perbenzoic acid,4-nonylamino-4-oxoperoxybutyric acid and diperoxydodecanedioic acid.Such bleaching agents are disclosed in U.S. Pat. No. 4,483,781, U.S.Pat. No. 740,446, EP 0 133 354 and U.S. Pat. No. 4,412,934. Highlypreferred bleaching agents also include 6-nonylamino-6-oxoperoxycaproicacid as described in U.S. Pat. No. 4,634,551.

Another category of bleaching agents that can be used encompasses thehalogen bleaching agents. Examples of hypohalite bleaching agents, forexample, include trichloro isocyanuric acid and the sodium and potassiumdichloroisocyanurates and N-chloro and N-bromo alkane sulphonamides.Such materials are normally added at 0.5-10% by weight of the finishedproduct, preferably 1-5% by weight.

The hydrogen peroxide releasing agents can be used in combination withbleach activators such as tetra-acetylethylenediamine (TAED),nonanoyloxybenzenesulfonate (NOBS, described in U.S. Pat. No.4,412,934), 3,5-trimethyl-hexsanoloxybenzenesulfonate (ISONOBS,described in EP 120 591) or pentaacetylglucose (PAG), which areperhydrolyzed to form a peracid as the active bleaching species, leadingto improved bleaching effect. In addition, very suitable are the bleachactivators C8 (6-octanamido-caproyl) oxybenzene-sulfonate, C9(6-nonanamido caproyl) oxybenzenesulfonate and C10 (6-decanamidocaproyl) oxybenzenesulfonate or mixtures thereof. Also suitableactivators are acylated citrate esters such as disclosed in EPapplication no. 91870207.7.

Useful bleaching agents, including peroxyacids and bleaching systemscomprising bleach activators and peroxygen bleaching compounds for usein cleaning compositions according to the invention are described inapplication U.S. application Ser. No. 08/136,626.

The hydrogen peroxide may also be present by adding an enzymatic system(i.e., an enzyme and a substrate therefor) which is capable ofgeneration of hydrogen peroxide at the beginning or during the washingand/or rinsing process. Such enzymatic systems are disclosed in EP 0 537381.

Bleaching agents other than oxygen bleaching agents are also known inthe art and can be utilized herein. One type of non-oxygen bleachingagent of particular interest includes photoactivated bleaching agentssuch as the sulfonated zinc and/or aluminium phthalocyanines. Thesematerials can be deposited upon the substrate during the washingprocess. Upon irradiation with light, in the presence of oxygen, such asby hanging clothes out to dry in the daylight, the sulfonated zincphthalocyanine is activated and, consequently, the substrate isbleached. Preferred zinc phthalocyanine and a photoactivated bleachingprocess are described in U.S. Pat. No. 4,033,718. Typically, detergentcomposition will contain about 0.025% to about 1.25%, by weight, ofsulfonated zinc phthalocyanine.

Bleaching agents may also comprise a manganese catalyst. The manganesecatalyst may, e.g., be one of the compounds described in “Efficientmanganese catalysts for low-temperature bleaching”, Nature 369: 637-639(1994).

Suds suppressors: Another optional ingredient is a suds suppressor,exemplified by silicones, and silica-silicone mixtures. Silicones cangenerally be represented by alkylated polysiloxane materials, whilesilica is normally used in finely divided forms exemplified by silicaaerogels and xerogels and hydrophobic silicas of various types. Thesematerials can be incorporated as particulates, in which the sudssuppressor is advantageously releasably incorporated in a water-solubleor water dispersible, substantially non surface-active detergentimpermeable carrier. Alternatively the suds suppressor can be dissolvedor dispersed in a liquid carrier and applied by spraying on to one ormore of the other components.

A preferred silicone suds controlling agent is disclosed in U.S. Pat.No. 3,933,672. Other particularly useful suds suppressors are theself-emulsifying silicone suds suppressors, described in German PatentApplication DTOS 2,646,126. An example of such a compound is DC-544,commercially available form Dow Corning, which is a siloxane-glycolcopolymer. Especially preferred suds controlling agent are the sudssuppressor system comprising a mixture of silicone oils and2-alkyl-alkanols. Suitable 2-alkyl-alkanols are 2-butyl-octanol whichare commercially available under the trade name Isofol 12 R.

Such suds suppressor systems are described in EP 0 593 841.

Especially preferred silicone suds controlling agents are described inEP application no. 92201649.8. Said compositions can comprise asilicone/silica mixture in combination with fumed nonporous silica suchas Aerosil^(R).

The suds suppressors described above are normally employed at levels offrom 0.001% to 2% by weight of the composition, preferably from 0.01% to1% by weight.

Other components: Other components used in detergent compositions may beemployed such as soil-suspending agents, soil-releasing agents, opticalbrighteners, abrasives, bactericides, tarnish inhibitors, coloringagents, and/or encapsulated or nonencapsulated perfumes.

Especially suitable encapsulating materials are water soluble capsuleswhich consist of a matrix of polysaccharide and polyhydroxy compoundssuch as described in GB 1,464,616.

Other suitable water soluble encapsulating materials comprise dextrinsderived from ungelatinized starch acid esters of substituteddicarboxylic acids such as described in U.S. Pat. No. 3,455,838. Theseacid-ester dextrins are, preferably, prepared from such starches as waxymaize, waxy sorghum, sago, tapioca and potato. Suitable examples of saidencapsulation materials include N-Lok manufactured by National Starch.The N-Lok encapsulating material consists of a modified maize starch andglucose. The starch is modified by adding monofunctional substitutedgroups such as octenyl succinic acid anhydride.

Antiredeposition and soil suspension agents suitable herein includecellulose derivatives such as methylcellulose, carboxymethylcelluloseand hydroxyethylcellulose, and homo- or co-polymeric polycarboxylicacids or their salts. Polymers of this type include the polyacrylatesand maleic anhydride-acrylic acid copolymers previously mentioned asbuilders, as well as copolymers of maleic anhydride with ethylene,methylvinyl ether or methacrylic acid, the maleic anhydride constitutingat least 20 mole percent of the copolymer. These materials are normallyused at levels of from 0.5% to 10% by weight, more preferably form 0.75%to 8%, most preferably from 1% to 6% by weight of the composition.

Preferred optical brighteners are anionic in character, examples ofwhich are disodium4,4′-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino)stilbene-2:2′-disulphonate,disodium4,4′-bis-(2-morpholino-4-anilino-s-triazin-6-ylamino-stilbene-2:2′-disulphonate,disodium4,4′-bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2:2′-disulphonate,monosodium4′,4″-bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2-sulphonate,disodium4,4′-bis-(2-anilino-4-(N-methyl-N-2-hydroxyethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulphonate,disodium4,4′-bis-(4-phenyl-2,1,3-triazol-2-yl)-stilbene-2,2′-disulphonate,disodium4,4′-bis(2-anilino-4-(1-methyl-2-hydroxyethylamino)-s-triazin-6-ylami-no)stilbene-2,2′disulphonate,sodium 2-(stilbyl-4″-(naphtho-1′,2′:4,5)-1,2,3-triazole-2″-sulphonateand 4,4′-bis(2-sulphostyryl)biphenyl.

Other useful polymeric materials are the polyethylene glycols,particularly those of molecular weight 1000-10000, more particularly2000 to 8000 and most preferably about 4000. These are used at levels offrom 0.20% to 5% more preferably from 0.25% to 2.5% by weight. Thesepolymers and the previously mentioned homo- or co-polymericpoly-carboxylate salts are valuable for improving whiteness maintenance,fabric ash deposition, and cleaning performance on clay, proteinaceousand oxidizable soils in the presence of transition metal impurities.

Soil release agents useful in compositions of the present invention areconventionally copolymers or terpolymers of terephthalic acid withethylene glycol and/or propylene glycol units in various arrangements.Examples of such polymers are disclosed in U.S. Pat. Nos. 4,116,885 and4,711,730 and EP 0 272 033. A particular preferred polymer in accordancewith EP 0 272 033 has the formula:(CH₃(PEG)₄₃)_(0.75)(POH)_(0.25)[T-PO)_(2.8)(T-PEG)_(0.4)]T(POH)_(0.25)((PEG)₄₃CH₃)_(0.75)wherein PEG is —(OC₂H₄)O—, PO is (OC₃H₆O) and T is (pOOC₆H₄CO).

Also very useful are modified polyesters as random copolymers ofdimethyl terephthalate, dimethyl sulfoisophthalate, ethylene glycol and1,2-propanediol, the end groups consisting primarily of sulphobenzoateand secondarily of mono esters of ethylene glycol and/or1,2-propanediol. The target is to obtain a polymer capped at both end bysulphobenzoate groups, “primarily”, in the present context most of saidcopolymers herein will be endcapped by sulphobenzoate groups. However,some copolymers will be less than fully capped, and therefore their endgroups may consist of monoester of ethylene glycol and/or1,2-propanediol, thereof consist “secondarily” of such species.

The selected polyesters herein contain about 46% by weight of dimethylterephthalic acid, about 16% by weight of 1,2-propanediol, about 10% byweight ethylene glycol, about 13% by weight of dimethyl sulfobenzoicacid and about 15% by weight of sulfoisophthalic acid, and have amolecular weight of about 3.000. The polyesters and their method ofpreparation are described in detail in EP 311 342.

Softening agents: Fabric softening agents can also be incorporated intolaundry detergent compositions in accordance with the present invention.These agents may be inorganic or organic in type. Inorganic softeningagents are exemplified by the smectite clays disclosed in GB-A-1 400898and in U.S. Pat. No. 5,019,292. Organic fabric softening agents includethe water insoluble tertiary amines as disclosed in GB-A1 514 276 and EP0 011 340 and their combination with mono C₁₂-C₁₄ quaternary ammoniumsalts are disclosed in EP 0 026 528 and di-long-chain amides asdisclosed in EP 0 242 919. Other useful organic ingredients of fabricsoftening systems include high molecular weight polyethylene oxidematerials as disclosed in EP 0 299 575 and 0 313 146.

Levels of smectite clay are normally in the range from 5% to 15%, morepreferably from 8% to 12% by weight, with the material being added as adry mixed component to the remainder of the formulation. Organic fabricsoftening agents such as the water-insoluble tertiary amines or dilongchain amide materials are incorporated at levels of from 0.5% to 5% byweight, normally from 1% to 3% by weight whilst the high molecularweight polyethylene oxide materials and the water soluble cationicmaterials are added at levels of from 0.1% to 2%, normally from 0.15% to1.5% by weight. These materials are normally added to the spray driedportion of the composition, although in some instances it may be moreconvenient to add them as a dry mixed particulate, or spray them asmolten liquid on to other solid components of the composition.

Polymeric dye-transfer inhibiting agents: The detergent compositionsaccording to the present invention may also comprise from 0.001% to 10%,preferably from 0.01% to 2%, more preferably form 0.05% to 1% by weightof polymeric dye-transfer inhibiting agents. Said polymeric dye-transferinhibiting agents are normally incorporated into detergent compositionsin order to inhibit the transfer of dyes from colored fabrics ontofabrics washed therewith. These polymers have the ability of complexingor adsorbing the fugitive dyes washed out of dyed fabrics before thedyes have the opportunity to become attached to other articles in thewash.

Especially suitable polymeric dye-transfer inhibiting agents arepolyamine N-oxide polymers, copolymers of N-vinyl-pyrrolidone andN-vinylimidazole, polyvinylpyrrolidone polymers, polyvinyloxazolidonesand polyvinylimidazoles or mixtures thereof.

Addition of such polymers also enhances the performance of the enzymesaccording the invention.

The detergent composition according to the invention can be in liquid,paste, gels, bars or granular forms.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 (both to Novo Industri A/S) and mayoptionally be coated by methods known in the art. Examples of waxycoating materials are poly(ethylene oxide) products (polyethyleneglycol,PEG) with mean molecular weights of 1000 to 20000; ethoxylatednonylphenols having from 16 to 50 ethylene oxide units; ethoxylatedfatty alcohols in which the alcohol contains from 12 to 20 carbon atomsand in which there are 15 to 80 ethylene oxide units; fatty alcohols;fatty acids; and mono-, di- and triglycerides of fatty acids. Examplesof film-forming coating materials suitable for application by fluid bedtechniques are given in GB 1483591.

Granular compositions according to the present invention can also be in“compact form”, i.e., they may have a relatively higher density thanconventional granular detergents, i.e., form 550 to 950 g/l; in suchcase, the granular detergent compositions according to the presentinvention will contain a lower amount of “Inorganic filler salt”,compared to conventional granular detergents; typical filler salts arealkaline earth metal salts of sulphates and chlorides, typically sodiumsulphate; “Compact” detergent typically comprise not more than 10%filler salt. The liquid compositions according to the present inventioncan also be in “concentrated form”, in such case, the liquid detergentcompositions according to the present invention will contain a loweramount of water, compared to conventional liquid detergents. Typically,the water content of the concentrated liquid detergent is less than 30%,more preferably less than 20%, most preferably less than 10% by weightof the detergent compositions.

The compositions of the invention may for example, be formulated as handand machine laundry detergent compositions including laundry additivecompositions and compositions suitable for use in the pretreatment ofstained fabrics, rinse added fabric softener compositions, andcompositions for use in general household hard surface cleaningoperations and dishwashing operations.

The following examples are meant to exemplify compositions for thepresent invention, but are not necessarily meant to limit or otherwisedefine the scope of the invention.

In the detergent compositions, the abbreviated component identificationshave the following meanings:

-   LAS: Sodium linear C₁₂ alkyl benzene sulphonate-   TAS: Sodium tallow alkyl sulphate-   XYAS: Sodium C_(1X)-C_(1Y) alkyl sulfate-   SS: Secondary soap surfactant of formula 2-butyl octanoic acid-   25EY: A C₁₂-C₁₅ predominantly linear primary alcohol condensed with    an average of Y moles of ethylene oxide-   45EY: A C₁₄-C₁₅ predominantly linear primary alcohol condensed with    an average of Y moles of ethylene oxide-   XYEZS: C_(1X)-C_(1Y) sodium alkyl sulfate condensed with an average    of Z moles of ethylene oxide per mole-   Nonionic: C₁₃-C₁₅ mixed ethoxylated/propoxylated fatty alcohol with    an average degree of ethoxylation of 3.8 and an average degree of    propoxylation of 4.5 sold under the tradename Plurafax LF404 by BASF    Gmbh-   CFAA: C₁₂-C₁₄ alkyl N-methyl glucamide-   TFAA: C₁₆-C₁₈ alkyl N-methyl glucamide-   Silicate: Amorphous Sodium Silicate (SiO₂:Na₂O ratio=2.0)-   NaSKS-6: Crystalline layered silicate of formula δ-Na₂Si₂O₅-   Carbonate: Anhydrous sodium carbonate-   Phosphate: Sodium tripolyphosphate-   MA/AA: Copolymer of 1:4 maleic/acrylic acid, average molecular    weight about 80,000-   Polyacrylate: Polyacrylate homopolymer with an average molecular    weight of 8,000 sold under the tradename PA30 by BASF Gmbh-   Zeolite A: Hydrated Sodium Aluminosilicate of formula    Na₁₂(AlO₂SiO₂)₁₂.27H₂O having a primary particle size in the range    from 1 to 10 micrometers-   Citrate: Tri-sodium citrate dihydrate-   Citric: Citric Acid-   Perborate: Anhydrous sodium perborate monohydrate bleach, empirical    formula NaBO₂.H₂O₂-   PB4: Anhydrous sodium perborate tetrahydrate-   Percarbonate: Anhydrous sodium percarbonate bleach of empirical    formula 2Na₂CO₃.3H₂O₂-   TAED: Tetraacetyl ethylene diamine-   CMC: Sodium carboxymethyl cellulose-   DETPMP: Diethylene triamine penta (methylene phosphonic acid),    marketed by Monsanto under the Tradename Dequest 2060-   PVP: Polyvinylpyrrolidone polymer-   EDDS: Ethylenediamine-N,N′-disuccinic acid, [S,S] isomer in the form    of the sodium salt-   Suds 25% paraffin wax Mpt 50° C., 17% hydrophobic silica, 58%    suppressor: paraffin oil-   Granular Suds: 12% Silicone/silica, 18% stearyl alcohol, 70%    suppressor: starch in granular form-   Sulphate: Anhydrous sodium sulphate-   HMWPEO: High molecular weight polyethylene oxide-   TAE 25: Tallow alcohol ethoxylate (25)

DETERGENT EXAMPLE I

A granular fabric cleaning composition in accordance with the inventionmay be prepared as follows:

Sodium linear C₁₂ alkyl 6.5 benzene sulfonate Sodium sulfate 15.0Zeolite A 26.0 Sodium nitrilotriacetate 5.0 Enzyme of the invention 0.1PVP 0.5 TAED 3.0 Boric acid 4.0 Perborate 18.0 Phenol sulphonate 0.1Minors Up to 100

DETERGENT EXAMPLE II

A compact granular fabric cleaning composition (density 800 g/l) inaccordance with the invention may be prepared as follows:

45AS 8.0 25E3S 2.0 25E5 3.0 25E3 3.0 TFAA 2.5 Zeolite A 17.0 NaSKS-612.0 Citric acid 3.0 Carbonate 7.0 MA/AA 5.0 CMC 0.4 Enzyme of theinvention 0.1 TAED 6.0 Percarbonate 22.0 EDDS 0.3 Granular sudssuppressor 3.5 water/minors Up to 100%

DETERGENT EXAMPLE III

Granular fabric cleaning compositions in accordance with the inventionwhich are especially useful in the laundering of colored fabrics wereprepared as follows:

LAS 10.7 — TAS 2.4 — TFAA — 4.0 45AS 3.1 10.0 45E7 4.0 — 25E3S — 3.068E11 1.8 — 25E5 — 8.0 Citrate 15.0 7.0 Carbonate — 10 Citric acid 2.53.0 Zeolite A 32.1 25.0 Na-SKS-6 — 9.0 MA/AA 5.0 5.0 DETPMP 0.2 0.8Enzyme of the invention 0.10 0.05 Silicate 2.5 — Sulphate 5.2 3.0 PVP0.5 — Poly (4-vinylpyridine)-N- — 0.2 Oxide/copolymer of vinyl-imidazole and vinyl- pyrrolidone Perborate 1.0 — Phenol sulfonate 0.2 —Water/Minors Up to 100%

DETERGENT EXAMPLE IV

Granular fabric cleaning compositions in accordance with the inventionwhich provide “Softening through the wash” capability may be prepared asfollows:

45AS — 10.0 LAS 7.6 — 68AS 1.3 — 45E7 4.0 — 25E3 — 5.0Coco-alkyl-dimethyl hydroxy- 1.4 1.0 ethyl ammonium chloride Citrate 5.03.0 Na-SKS-6 — 11.0 Zeolite A 15.0 15.0 MA/AA 4.0 4.0 DETPMP 0.4 0.4Perborate 15.0 — Percarbonate — 15.0 TAED 5.0 5.0 Smectite clay 10.010.0 HMWPEO — 0.1 Enzyme of the invention 0.10 0.05 Silicate 3.0 5.0Carbonate 10.0 10.0 Granular suds suppressor 1.0 4.0 CMC 0.2 0.1Water/Minors Up to 100%

DETERGENT EXAMPLE V

Heavy duty liquid fabric cleaning compositions in accordance with theinvention may be prepared as follows:

I II LAS acid form — 25.0 Citric acid 5.0 2.0 25AS acid form 8.0 —25AE2S acid form 3.0 — 25AE7 8.0 — CFAA 5.0 — DETPMP 1.0 1.0 Fatty acid8.0 — Oleic acid — 1.0 Ethanol 4.0 6.0 Propanediol 2.0 6.0 Enzyme of theinvention 0.10 0.05 Coco-alkyl dimethyl — 3.0 hydroxy ethyl ammoniumchloride Smectite clay — 5.0 PVP 2.0 — Water/Minors Up to 100%Leather Industry Applications

A subtilase of the invention may be used in the leather industry, inparticular for use in depilation of skins.

In said application a subtilase variant of the invention is preferablyused in an enzyme composition which further comprises another protease.

For a more detailed description of suitable other proteases, see sectionrelating to suitable enzymes for use in a detergent composition.

Wool lndustry Applications

A subtilase of the invention may be used in the wool industry, inparticular for use in cleaning of clothes comprising wool.

In said application a subtilase variant of the invention is preferablyused in an enzyme composition which further comprises another protease.

For a more detailed description of suitable other proteases, see sectionrelating to suitable enzymes for use in a detergent composition.

The invention is described in further detail in the following exampleswhich are not in any way intended to limit the scope of the invention asclaimed.

Materials And Methods

Strains:

B. subtilis DN1885 (Diderichsen et al., 1990).

B. lentus 309 and 147 are specific strains of Bacillus lentus, depositedwith the NCIB and accorded the accession numbers NCIB 10309 and 10147,and described in U.S. Pat. No. 3,723,250 incorporated by referenceherein.

E. coliMC 1000 (M. J. Casadaban and S. N. Cohen (1980); J. Mol. Biol.138 179-207), was made r⁻,m⁺ by conventional methods and is alsodescribed in U.S. application no. 039,298.

Plasmids:

pJS3: E. coli-B. subtilis shuttle vector containing a synthetic geneencoding for subtilase 309. (Described by Jacob Schiødt et al. inProtein and Peptide letters 3:39-44 (1996)).

pSX222: B. subtilis expression vector (Described in WO 96/34946).

General Molecular Biology Methods:

Unless otherwise mentioned the DNA manipulations and transformationswere performed using standard methods of molecular biology (Sambrook etal., Molecular cloning: A laboratory manual, Cold Spring Harbor Lab.,Cold Spring Harbor, N.Y. (1989); Ausubel, F. M. et al. (eds.) “CurrentProtocols in Molecular Biology”. John Wiley and Sons, 1995; Harwood, C.R., and Cutting, S. M. (eds.) “Molecular Biological Methods forBacillus”. John Wiley and Sons, 1990).

Enzymes for DNA manipulations were used according to the specificationsof the suppliers.

Enzymes for DNA Manipulations

Unless otherwise mentioned all enzymes for DNA manipulations, such asrestiction endonucleases, ligases etc., are obtained from New EnglandBiolabs, Inc.

Proteolytic Activity

In the context of this invention proteolytic activity is expressed inKilo NOVO Protease Units (KNPU). The activity is determined relativelyto an enzyme standard (SAVINASEÔ), and the determination is based on thedigestion of a dimethyl casein (DMC) solution by the proteolytic enzymeat standard conditions, i.e., 50° C., pH 8.3, 9 min. reaction time, 3min. measuring time. A folder AF 220/1 is available upon request to NovoNordisk A/S, Denmark, which folder is hereby included by reference.

A GU is a Glycine Unit, defined as the proteolytic enzyme activitywhich, under standard conditions, during a fifteen minute incubation at40° C., with N-acetyl casein as the substrate, produces an amount ofNH₂-group equivalent to 1 mmole of glycine.

Enzyme activity can also be measured using the PNA assay, according tothe reaction with the soluble substratesuccinyl-alanine-alanine-proline-phenyl-alanine-para-nitrophenol, whichis described in the Journal of American Oil Chemists Society, Rothgeb,T. M., Goodlander, B. D., Garrison, P. H., and Smith, L. A. (1988).

Fermentation:

Fermentation of subtilase enzymes was performed at 30° C. on a rotaryshaking table (300 r.p.m.) in 500 ml baffled Erlenmeyer flaskscontaining 100 ml BPX medium for 5 days.

Consequently in order to make a 2 liter broth, 20 Erlenmeyer flasks werefermented simultaneously.

Media: BPX: Composition (per liter) Potato starch 100 g Ground barley 50g Soybean flour 20 g Na₂HPO₄ × 12 H₂O 9 g Pluronic 0.1 g Sodiumcaseinate 10 g

The starch in the medium is liquefied with alpha-amylase and the mediumis sterilized by heating at 120° C. for 45 minutes. After sterilizationthe pH of the medium is adjusted to 9 by addition of NaHCO₃ to 0.1 M.

EXAMPLES Example 1

Construction and Expression of Enzyme Variants

Site-Directed Mutagenesis:

Subtilase 309 site-directed variants were made by the “Unique siteelimination (USE)” or the “Uracil-USE” technique described respectivelyby Deng et al. (Anal. Biochem. 200: 81-88 (1992)) and Markvardsen et al.(BioTechniques 18(3): 371-372 (1995)).

The template plasmid was pJS3, or an analogue thereof containing avariant of subtilase 309, e.g., USE mutagenesis was performed on pJS3analogue containing a gene encoding the N252L variant with aoligonucleotide directed to the construct of a T255I variant resultingin a final N252L+T255I subtilase 309 variant.

The in pJS3 constructed subtilase 309 variants were then subcloned intothe B. subtilis pSX222 expression plasmid, using the restriction enzymesKpnI and MluI.

Localized Random Mutagenesis:

The overall strategy used to perform localized random mutagenesis was:

A mutagenic primer (oligonucleotide) was synthesized which correspondsto the part of the DNA sequence to be mutagenized except for thenucleotide(s) corresponding to amino acid codon(s) to be mutagenized.

Subsequently, the resulting mutagenic primer was used in a PCR reactionwith a suitable opposite primer. The resulting PCR fragment was purifiedand digested and cloned into an E. coli-B. subtilis shuttle vector.

Alternatively and if necessary, the resulting PCR fragment was used in asecond PCR reaction as a primer with a second suitable opposite primerso as to allow digestion and cloning of the mutagenized region into theshuttle vector. The PCR reactions were performed under normalconditions.

Following this strategy a localized random library was constructed inSAVINASE wherein positions N252, T255 and S259 were completelyrandomized.

One oligonucleotide was synthesized with 25% of each of the four bases(N) in the first and the second base at amino acid codons wanted to bemutagenized. The third nucleotide (the wobble base) in codons weresynthesized with 50% G/50% C(S) to avoid two (TM, TGA) of the three stopcodons.

The mutagenic primer (5′-C TTC TGC GTT MC MG TCC GCT TCC ATA CM GTT CGTSNN TCC TM ACT SNN TGC CGT SNN CTT TAG ATG ATT-3′ (anti-sense) (SEQ IDNO: 11)) was used in a PCR reaction with a suitable opposite primer(e.g., 5′-GM CTC GAT CCA CG ATT TC-3′ (sense) (SEQ ID NO: 12)) and theplasmid pJS3 as the template. The resulting PCR product was cloned intothe pJS3 shuttle vector by using the restriction enzymes XhoI and HpaI.

The in pJS3 constructed localized random library was then subcloned intothe B. subtilis SX222 expression plasmid, using the restriction enzymesKpnI and MluI.

The library prepared contained approximately 100,000 individualclones/library.

Ten randomly chosen colonies were sequenced to confirm the mutationsdesigned.

In order to purify a subtilase variant of the invention, the B. subtilispSX222 expression plasmid comprising a variant of the invention wastransformed into a competent B. subtilis strain and was fermented asdescribed above in a medium containing 10 micrograms/ml Chloramphenicol(CAM).

Example 2

Purification of Enzyme Variants

This procedure relates to purification of a 2 liter scale fermentationof subtilisin 147, subtilisin 309 or mutants thereof.

Approximately 1.6 liters of fermentation broth were centrifuged at 5000rpm for 35 minutes in 1 liter beakers. The supernatants were adjusted topH 6.5 using 10% acetic acid and filtered on Seitz Supra S100 filterplates.

The filtrates were concentrated to approximately 400 ml using an AmiconCH2A UF unit equipped with an Amicon S1Y10 UF cartridge. The UFconcentrate was centrifuged and filtered prior to absorption at roomtemperature on a Bacitracin affinity column at pH 7. The protease waseluted from the Bacitracin column at room temperature using 25%2-propanol and 1 M sodium chloride in a buffer solution with 0.01dimethylglutaric acid, 0.1 M boric acid and 0.002 M calcium chlorideadjusted to pH 7.

The fractions with protease activity from the Bacitracin purificationstep were combined and applied to a 750 ml Sephadex G25 column (5 cmdia.) equilibrated with a buffer containing 0.01 M dimethylglutaricacid, 0.2 M boric acid and 0.002 M calcium chloride adjusted to pH 6.5.

Fractions with proteolytic activity from the Sephadex G25 column werecombined and applied to a 150 ml CM Sepharose CL 6B cation exchangecolumn (5 cm dia.) equilibrated with a buffer containing 0.01 Mdimethylglutaric acid, 0.2 M boric acid, and 0.002 M calcium chlorideadjusted to pH 6.5.

The protease was eluted using a linear gradient of 0-0.1 M sodiumchloride in 2 liters of the same buffer (0-0.2 M sodium chloride in caseof subtilisin 147).

In a final purification step, fractions from the CM Sepharose columncontaining protease were combined and concentrated in an Amiconultrafiltration cell equipped with a GR81PP membrane (from the DanishSugar Factories Inc.).

By using the techniques of Example 1 for the construction and the aboveisolation procedure, the following subtilisin 309 variants were producedand isolated:

N252L+T255I

N252V+T255A

N252M+T255C+S259H

N252S+T255E+S259C; and

N252K+T255S+S259C.

Example 3

Wash Performance of Detergent Compositions Comprising Enzyme Variants

The following examples provide results from a number of washing teststhat were conducted under the following conditions:

Detergent Protease Model Detergent 95 Detergent dose 3.0 g/l pH 10.5Wash time 15 min. Temperature 15° C. Water hardness 6° dH EnzymesSubtilisin 309 variants as listed below Enzyme conc. 10 nM Test system150 ml glass beakers with a stirring rod Textile/volume 5 textile pieces(Ø 2.5 cm) in 50 ml detergent Test material EMPA117 from Center forTestmaterials, Holland

The detergent used is a simple model formulation. The pH was adjusted to10.5 which is within the normal range for a powder detergent. Thecomposition of model detergent 95 was as follows:

25% STP (Na₅P₃O₁₀) 25% Na₂SO₄ 10% Na₂CO₃ 20% LAS (Nansa 80S) 5.0% Nonionic tenside (Dobanol 25-7) 5.0%  Na₂Si₂O₅ 0.5% Carboxymethylcellulose (CMC) 9.5%  Water

Water hardness was adjusted by adding CaCl₂ and MgCl₂ (Ca²⁺:Mg²⁺=2:1) todeionized water (see also Surfactants in Consumer Products—Theory,Technology and Application, Springer Verlag 1986). The pH of thedetergent solution was adjusted to pH 10.5 by addition of HCl.

Measurement of reflectance (R) on the test material was done at 460 nmusing a Macbeth ColorEye 7000 photometer (Macbeth, Division ofKollmorgen Instruments Corporation, Germany). The measurements were doneaccording to the manufacturer's protocol.

The wash performance of the subtilisin 309 variants was evaluated bycalculating a performance factor:P=(R _(Variant) −R _(Blank))/(R _(Savinase) −R _(Blank))wherein

P: Performance factor

R_(variant): Reflectance of test material washed with variant

R_(savinase): Reflectance of test material washed with Savinase®

R_(Blank): Reflectance of test material washed with no enzyme

The subtilisin 309 variants all have improved wash performance comparedto Savinase®—i.e., P>1.

The variants are divided into improvement classes designated withcapital letters:

Class A: 1<P≦1.5

Class B: 1.5<P≦2

Class C: P>2

TABLE IV Subtilisin 309 variants and improvement classes. Improvementclass Variants A N252L + T255I N252V + T255A N252M + T255C + S259HN252S + T255E + S259C N252K + T255S + S259C B C

1. A modified subtilase, comprising a modification which is asubstitution of the amino acid at position 252 with K or L, wherein theposition is numbered according to the amino acid sequence of the maturesubtilisin BPN′ set forth in SEQ ID NO:1.
 2. The modified subtilase ofclaim 1, which comprises a modification which is a substitution of theamino acid at position 252 with K.
 3. The modified subtilase of claim 1which comprises a modification which is a substitution of the amino acidat position 252 with L.
 4. The modified subtilase of claim 1, whichfurther comprises a modification which is a substitution of the aminoacid at position 255 with A or E.
 5. The modified subtilase of claim 1,which comprises a modification which is a substitution of the amino acidat position 252 with L and a modification which is a substitution of theamino acid at position 255 with A.
 6. The modified subtilase of claim 1,comprising at least one further modification which is an amino acidsubstitution or insertion at one or more positions selected from thegroup consisting of 27, 36, 57, 76, 97, 101, 104, 120, 123, 167, 170,206, 218, 222, 224, 235 and
 274. 7. The modified subtilase of claim 6,wherein the at least one further modification is selected from the groupconsisting of K27R, *36D, S57P, N76D, G97N, S101G, V104A, V104N, V104Y,H102D, N123S, Y167A, Y167I, R170L, R170N, R170S, Q206E, N218S, M222A,M222S, T224S, K235L, and T274A.
 8. The modified subtilase of claim 7,wherein the at least one further modification is selected from the groupconsisting of K27R+V104Y+N123S+T274A, N76D+V104A, V104N+S101G, and anyother combination of K27R, N76D, S101G, V104A, V104N, V104Y, N123S, andT274A.
 9. The modified subtilase of claim 1, comprising at least onefurther modification which is an amino acid substitution at one or morepositions selected from the group consisting of 129, 131, 133 and 194.10. The modified subtilase of claim 9, wherein the at least one furthermodification is selected from the group consisting of P129K, P131H,A133D, A133P, and A194P.
 11. The modified subtilase of claim 1, whereinthe subtilase is a sub-group I-S1 subtilase.
 12. The modified subtilaseof claim 11, wherein the subtilase is selected from the group consistingof subtilisin I168, subtilisin BPN′, subtilisin DY, and subtilisinGarlsberg.
 13. The modified subtilase of claim 1, wherein the subtilaseis a sub-group I-S2 subtilase.
 14. The modified subtilase of claim 13,wherein the subtilase is selected from the group consisting ofsubtilisin 147, subtilisin 309, subtilisin PB92, and subtilsin YaB. 15.A composition comprising a modified subtilase of claim 1 and asurfactant.
 16. The composition of claim 15, which additionallycomprises an amylase, cellulase, cutinase, lipase, oxidoreductase, oranother protease.